*All reagents
should be thawed quickly and then stored on ice.
**Click here to read about the importance of magnesium concentration!!

Do
it yourself from invidual components

In order to do PCR(polymerase
chain reaction), you need the following components:

template DNA

reaction buffer with the appropriate amount of
MgCl2

dNTPs

forward primer

reverse primer

Taq DNA polymerase

The question is, how much of each? Traditionally,
PCR is done in 100 μl volumes and the reaction buffer comes as a 10X
stock solution (meaning it is 10 times too concentrated and must be diluted).
For our labs, the dNTPs are in a 20X stock solution; the primers and template
are in 100X stock solutions; and the Taq is in a 200X stock solution. Therefore,
you should set up a table, fill in the blanks, and add the reagents in the
order presented below (always protect your enzymes by adding them last -
they are touchy creatures and expensive).
Once you have your mixture ready, put it in the thermal cycler and start
the process. We will be using the program called IDH with the heated lid
ENABLED.

IDH Temperature Cycle:

Step 1: 95° C
5 minutes (denature template)

Step 2: 95° C 1 minute
(denature dsDNA)

Step 3: 55° C 1 minute
(Tm minus 5 degrees)

Step 4: 72° C 1 minute
(amplify about 1 kb per minute)

Step 5: Repeat Steps 2 through 4, 29 more
times

Step 6: Store at RT

REAGENT*

VOLUME (μl)

FINAL CONC.

water

N/A

10x buffer with magnesium**

1X

or buffer w/o Mg

1X

plus 25 mM MgCl2 stock

varies (often 1.5 mM)

template DNA (~1 ng gDNA)

1 ng

dNTPs (4 mM stock for each base)

(200μM stock for each base)

primer #1

1 μM

primer #2

1 μM

Taq* (500 units/1 μL stock)

2.5 units

FINAL VOLUME

100

*The Taq must be kept in the special "cold block" in
order to keep the enzyme as cold as possible while it is being pipetted.
All other reagents should be thawed quickly and then stored on ice.

**Click here to read about the importance of magnesium concentration!!